The proposed research is to elucidate the molecular basis of the facilitated sodium entry mechanism which exists in the outer (or apical) membrane of all tight epithelial tissues. This sodium entry step is an essential component of the overall active transport system responsible for the net movement of salt and water across these structures. This entry process is passive, and is sensitive to inhibition by the diuretic drug, amiloride. The analysis of the mechanism of Na entry will encompass determinations of cation selectivities, the effects of chemical group-specific reagents and other pharmacological substances on Na entry and amiloride efficacy, a determination of site number from binding studies of a photosensitive amiloride analogue (bromoamiloride), and the preparation of apical membrane vesicles containing functional Na-entry sites. A comparative approach, utilizing the isolated amphibian skin and bladder preparations as well as the isolated rabbit colon, will be taken. These studies will contribute to our knowledge of the kinetic and biochemical properties of eipthelial transport systems and increase our comprehension of the mode of action of amiloride and other diuretic compounds.